Widespread antibiotic use continues to be a public health problem worldwide. In addition to driving development of antibiotic resistance, another consequence of antibiotic use has been an increase in Clostridium difficile infection (CDI), now the most common cause of nosocomial diarrhea. CDI results in significant morbidity and mortality with over 500,000 cases and 14,000 deaths due to CDI annually in the US alone. Antibiotics administered for other infections decrease the diversity of the normal gut microbiota resulting in a loss of gut colonization resistance which enables C. difficile, a toxin-producing anaerobic pathogen resistant to most antibiotics, to proliferate without having to compete with normal intestinal microbes. The microbial ecosystem of the intestine plays a critical protective role against CDI, well-illustrated by the high success rate reported for fecal microbiota transplantation (FMT) for the treatment of recurrent CDI. However, while very successful, FMT is not a palatable procedure, concerns exist about donor transmission of infection, and mechanisms of action remain poorly understood. To address these issues, we developed a defined Microbial Ecosystem Therapeutic (MET-1), prototype that we have already used successfully to cure two patients with recurrent C. difficile infection (CDI) that was refractory t all other antibiotic treatment. Consistent with the concept of a core microbiome that encompasses key functions required for normal intestinal homeostasis, Microbial Ecosystem Therapeutics, is a means of replacing a dysfunctional, damaged ecosystem with a healthy ecosystem. Now that we have demonstrated feasibility of the MET approach, the current application proposes to extend our studies, using novel methods of rational design, to develop new MET formulations to treat C. difficile infections caused by hypervirulent strains that do not respond to traditional antibiotics. We hypothesize that utilization of selected microbial communities will be effective at protecting the host against CDI. These formulations can be designed to be resilient enough to withstand further antibiotic insults and additional CDI relapse. Our overall objective is to optimize MET formulations based on rational design, and to determine the mechanisms whereby MET decreases the incidence and severity of CDI In the R21 phase, we will develop new and improved MET formulations to treat CDI, using novel methods of rational design (Aim 1). Both the in vitro and in vivo readouts will be employed to test efficacy of the new MET formulations and to further investigate mechanism (Aim 2), using our MET-1 prototype as a comparator control. Once the defined milestones of the R21 grant are met, we plan to transition to an R33 phase to further develop the MET product into a FDA approved commercial product for the treatment of recurrent CDI. The focus of the R33 phase will be to create reliable manufacturing processes (Aim 3) and quality control systems (Aim 4) to ensure that the product being manufactured is consistent from batch to batch and to ensure that we have a product that will meet the standards required to submit an IND.